Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.
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PMID:Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin. 300 81

The endothelial cell-type plasminogen activator inhibitor (PAI-1) may exist in an inactive, latent form that can be converted into an active form upon treatment of the protein with denaturants, such as sodium dodecyl sulfate, guanidine HCl, or urea. The present paper demonstrates that latent PAI-1 can be activated by lipid vesicles containing the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol. The presence of a net negative charge on the phospholipid headgroup is essential for activation, since lipid vesicles consisting exclusively of zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, do not activate PAI-1. In the presence of PS vesicles, PAI-1 inhibited tissue-type plasminogen activator 50-fold more effectively than in the absence of phospholipids, whereas sodium dodecyl sulfate enhanced PAI-1 activity by 25-fold. In mixed phospholipid vesicles containing PS and phosphatidylcholine in various molar ratios, the extent of PAI-1 activation was directly related to the PS content of the phospholipid membrane. Ca2+ ions interfered with the inhibitory activity of PS-activated PAI-1, suggesting that Ca2+ ions may regulate PAI-1 activity in the presence of negatively charged phospholipids. An important consequence of these findings is that, as in blood coagulation, negatively charged phospholipids may play an important regulatory role in controlling the fibrinolytic system by activating an inhibitor of tissue-type plasminogen activator.
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PMID:Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids. 312 98

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45 degrees C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.
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PMID:Immunoaffinity purification of HTC rat hepatoma cell plasminogen activator-inhibitor-1. 312 13

Native one-chain tissue plasminogen activator (t-PA) was rapidly converted to the two-chain form by trypsin-Sepharose cleavage. This caused an increase in the amidolytic activity on low molecular weight peptide substrates, while plasminogen activation in the presence of fibrin markedly decreased. Cleavage sites were identified by N-terminal sequence analysis of reduced and carboxymethylated peptides. In the B-chain, the expected cleavage at Arg278-Ile279 was identified. Furthermore, a specific cleavage site was found at Arg302-Ser303, 24 amino acids from the N-terminus of the B-chain. The peptide released by this cleavage (designated B1-24) remained associated with the activator molecule by strong noncovalent interactions but could be dissociated under denaturing conditions (4 mol/L of guanidine hydrochloride), leading to a 20-fold decrease in amidolytic activity. Addition of purified B1-24 peptide to t-PA treated in this manner restored the activity in a concentration-dependent way. In contrast to trypsin, cleavage of the single-chain t-PA molecule with endoproteinase Lys-C generated a two-chain form of the activator, without simultaneous increase in the amidolytic activity. By sequence analysis, a major cleavage was identified at Lys280-Gly281, two residues into the B-chain. Together, the results presented provide additional information on the one-chain to two-chain conversion of t-PA and the role of the free N-terminus of the B-chain.
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PMID:Proteolytic modification of tissue plasminogen activator: importance of the N-terminal part of the catalytically active B-chain for enzymatic activity. 314 3

Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.
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PMID:Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. 657 65

The experiments reported here were carried out to define in greater detail actin's stimulation of plasmin generation by t-PA. Actin did not alter t-PA's hydrolysis of a synthetic substrate, and thus is unlikely to have a direct effect upon t-PA's proteolytic activity. When studied in a single-stage assay, actin accelerated t-PA-mediated plasmin generation from both Glu-plasminogen and Lys-plasminogen, indicating the central role of ternary complex formation. Although actin does not appear to bind two-chain urokinase (tcu-PA), it stimulates tcu-PA's cleavage of Glu-plasminogen. This finding suggests that actin alters the conformation of Glu-plasminogen to an open form. The failure of actin to increased plasmin generation by tcu-PA acting on Lys-plasminogen, which is in an open configuration, is consistent with this interpretation. Immunoglobin G, which shares with actin the property of binding to Glu-plasminogen after nicking by plasmin, did not stimulate tcu-PA's cleavage of Glu-plasminogen, indicating the uniqueness of actin's effects and suggesting interactions between actin and plasminogen at multiple binding sites. Unlike fibrin and heparin, whose stimulation of t-PA is related to polymer length actin is able to stimulate t-PA when presented in either a monomeric or polymeric form. Denaturation of actin by exposure to urea and guanidine increased its ability to stimulate plasmin generation by t-PA. Because actin's structure is maintained by a noncovalently bound adenine nucleotide (ATP or ADP), exposure to ATP/ADPases found in plasma and on cell membranes might also result in its denaturation. Actin treated with an enzyme functionally similar to such ecto-ATP/ADPases, potato apyrase, was more potent than native actin in stimulating plasmin generation by t-PA. The effects of apyrase were blocked by the addition of the plasma actin-binding proteins, gelsolin and the vitamin D-binding protein (DBP). Thus, denaturation of actin may occur in under physiologic conditions, with potential biological consequences. Actin thus appears to be unique with regard to its interactions with the fibrinolytic system and plasma actin-binding proteins may serve to protect the host from the effects of denatured actin.
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PMID:Actin stimulates plasmin generation by tissue and urokinase-type plasminogen activators. 823 51

Recent studies indicate that 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) and 24R,25-dihydroxyvitamim D3 (24R,25[OH]2D3) differentially regulate proliferation, differentiation, and matrix synthesis of growth plate chondrocytes. To determine whether both metabolites play the same or different roles in vivo, we used the vitamin D-deficient rat as a model. Rickets was induced and then reversed by administering a single dose of ergocalciferol, 1alpha,25(OH)2D3, or 24R,25(OH)2D3 and euthanizing the animals after 4, 24, 48, or 72 h. Growth plates were either processed for histology and histomorphometry or extracted with buffered guanidine-HCl. Neutral metalloproteinase activity in the extracts was measured by use of aggrecan-containing beads, and collagenase activity was determined by use of radioactive type I collagen. The levels of tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator were also determined. The morphology of the growth plate varied as a function of treatment. While 24R,25(OH)2D3 appeared to affect cell maturation and 1alpha,25(OH)2D3 appeared to affect terminal differentiation and calcification, response to ergocalciferol was indicative of the combined responses to the individual metabolites. Enzyme activity was regulated in a differential manner. Treatment with ergocalciferol produced a rapid decline in both neutral metalloproteinase and collagenase activities that was statistically significant by 4 h. By contrast, 1alpha,25(OH)2D3 had no effect on neutral metalloproteinase activity but caused a significant decrease in both active and total collagenase activity by 4 h, while 24R,25(OH)2D3 decreased neutral metalloproteinase activity by 48 h and had no effect on collagenase activity. Ergocalciferol had no effect on TIMP levels at any time examined, whereas 1alpha,25(OH)2D3 caused an increase at 48 and 72 h and 24R,25(OH)2D3 completely blocked TIMP production at 4 and 24 h. By contrast, plasminogen activator activity by ergocalciferol was decreased at 4 h, increased by 1alpha,25(OH)2D3 at 4 and 24 h, and decreased by 24R,25(OH)2D3 at all time points examined. These in vivo results confirm our previous cell culture observations showing that growth plate chondrocytes are differentially regulated by 1alpha,25(OH)2D3 and 24R,25(OH)2D3. Moreover, they show definitively that these two vitamin D metabolites play distinct roles not only in regulating neutral metalloproteinase and collagenase activities in growth plate cartilage but in cell maturation and calcification of this tissue in vivo.
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PMID:Effect of 1alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 on metalloproteinase activity and cell maturation in growth plate cartilage in vivo. 1144 27

Transforming growth factor beta-1 (TGF-beta1) is secreted in a biologically inactive form and stored in the extracellular matrix as a 290 kDa complex consisting of the mature TGF-beta1 homodimer (Mr 25 kDa), the latency-associated peptide (LAP; Mr 75 kDa), and the latent TGF-beta1 binding protein-1 (LTBP1; Mr 190 kDa). Latent TGF-beta1, composed of these three components, is known as the "large latent TGF-beta1 complex." In contrast, latent TGF-beta1 without LTBP1 is known as "small latent TGF-beta1." For all latent forms, dissociation of the TGF-beta1 homodimer from LAP is necessary for growth factor activation and acquisition of biological activity. Matrix vesicles produced by growth plate chondrocytes contain matrix metalloproteinases that can activate small latent TGF-beta1. The enzyme responsible for this is matrix metalloproteinase-3 (MMP-3), although matrix vesicles also contain MMP-2 and plasminogen activator. The present study tested the hypothesis that matrix vesicle enzymes are also involved in the release of the large latent TGF-beta1 complex stored in the extracellular matrix. Matrix vesicles were isolated from cultures of resting zone and growth zone chondrocytes and metalloproteinases present in the matrix vesicles extracted with guanidine-HCl. Chondrocyte extracellular matrices were prepared by lysing confluent cultures and removing the lysed cells. The matrices were incubated with matrix vesicle extracts and the release of total and active TGF-beta1 was determined. To determine if MMP-2 or MMP-3 was involved in the release, matrix vesicle extracts were preincubated with anti-MMP-2 antibody or anti-MMP-3 antibody to selectively deplete the enzyme activity. Matrices were also treated with rhMMP-2 or rhMMP-3. To determine the identity of the released protein(s), digests were separated on SDS-polyacrylamide gels and Western blotting analysis was performed using a specific antibody to LTBP1. Matrix vesicle extracts released both active and total (=latent + active) TGF-beta1 in a time-dependent manner, with peak release after 1 hour of incubation. The amount of total TGF-beta1 released was 10 times higher than the release of active TGF-beta1. The effect of the matrix vesicle extracts was dose-dependent; in addition, the amount and ratio of active to total TGF-b1 released was very similar, irrespective of the source of matrix or matrix vesicle extracts. Pre-incubation of matrix vesicle extracts with anti-MMP-3 antibody blocked the release of active and total TGF-beta1, whereas pre-incubation with pre-immune IgG or anti-MMP-2 antibody had no effect. The addition of rhMMP-3, but not rhMMP-2, caused a dose-dependent increase in the release of total, but not active, TGF-beta1. Western analysis confirmed that both matrix vesicle extracts and rhMMP-3 released the large latent TGF-beta1 complex from the matrix. In addition to the expected 290, 230, and 190 kDa bands, samples run without reduction also contained proteins of molecular weights 110 and 50 kDa that reacted with the anti-LTBP1 antibody. When these same samples were electrophoresed after reduction, the high molecular weight immunoreactive bands disappeared and three bands of molecular weight 75, 32, and 25 kDa were observed. These results indicate that matrix vesicles contain enzymes, especially MMP-3, which are responsible for the release of TGF-beta1 from the matrix, most of which is in latent form. Further, the data suggest that release of the large complex occurs via cleavage at several novel sites in the 130 kDa LTBP1 molecule. Since matrix vesicle MMP-3 is also able to activate small latent TGF-beta1, these results suggest that the large latent TGF-beta1 complex protects against activation of the small latent TGF-beta1. Thus, the data suggest that release of the large latent TGF-bl complex from the matrix and activation of the latent growth factor are only two steps of what must be at least a three-step process.
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PMID:The first stage of transforming growth factor beta1 activation is release of the large latent complex from the extracellular matrix of growth plate chondrocytes by matrix vesicle stromelysin-1 (MMP-3). 1190 8

Bovine pancreatic PLA(2) (phospholipase A(2)) is a 14 kDa protein whose structure is highly cross-linked by seven disulphide bonds. We investigated the structural stability of this enzyme by the method of 'disulphide-scrambling' with denaturants such as urea, GdmCl (guanidine hydrochloride), GdmSCN (guanidine thiocyanate) and at high temperatures in the presence of 2-mercaptoethanol (0.2 mM) as thiol initiator. Reverse-phase HPLC was used to follow denaturation. To denature 50% of the native protein, 1.25 M GdmSCN, approx. 3 M GdmCl and higher than 8 M urea were required. Only 20% of the protein was denatured after 2 h at 60 degrees C, whereas complete denaturation was seen after 2 h at 70 degrees C and within 30 min at 80 degrees C. A distinct enhancement of stability was observed when denaturation was conducted in the presence of 10 mM calcium chloride, which has not been reported previously. CD studies of GdmCl denaturation of bovine PLA(2) showed that 2.5 M GdmCl was required to denature 50% of the protein in the presence of 0.2 mM 2-mercaptoethanol (in agreement with the HPLC analysis), whereas 6.4 M GdmCl was necessary to denature 50% of the protein in the absence of a thiol initiator. Conformational stability (Delta G (water)) was estimated to be 8.7 kcal/mol (1 cal=4.184 J) by 'disulphide-intact' denaturation (where 'native' disulphide framework was unaffected) and 2.5 kcal/mol by 'disulphide-scrambling' denaturation (involved breaking of native disulphides and formation of 'non-native' ones). The difference, Delta(Delta G (water)), of 6.2 kcal/mol was the conformational stability contributed by the 'native-framework' of seven disulphides. Using bovine PLA(2) as an example, we have demonstrated a novel comparative technique, where the conformational stability study of a disulphide-containing protein, with a common denaturant, in both the presence and absence of catalytic amounts of a thiol initiator can be used as a convenient method to estimate selectively and quantitatively the actual contribution of the 'native disulphide bond network' towards the global conformational stability of the protein.
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PMID:Investigating conformational stability of bovine pancreatic phospholipase A2: a novel concept in evaluating the contribution of the 'native-framework' of disulphides to the global conformational stability of proteins. 1453 80

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.
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PMID:A comparative investigation on different refolding strategies of recombinant human tissue-type plasminogen activator derivative. 1661 26


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